Symptoms of T-cell hyperactivation shape the course and outcome of HIV-1 infection, but the molecular mechanism(s) underlying this chronic immune activation are not well understood. We have identified a novel mechanism by which the HIV-1 Tat protein leads to chronic immune activation. We find that Tat hyperactivates T cells by blocking the deacetylase activity of SIRT1 (Kwon et al, Cell Host Microbe, 2008). SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase and an important regulator of the transcription factor NF-B. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate the RelA/p65 subunit of NF-B. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-B-responsive genes, such as interleukin-2 (IL-2). These results support a model where the normal function of SIRT1 as a negative regulator of T-cell activation is suppressed by Tat during HIV infection. We propose to define the novel role of SIRT1 as a regulator of immune activation in HIV-infected T cells. We have new and unpublished findings that link the SIRT1 deacetylase activity with immune-suppressive functions of regulatory T cells (Tregs). Special focus of this proposal lies therefore on the characterization of the function of Tat and SIRT1 in Tregs and on the identification of novel SIRT1 substrates in effector and regulatory T cells populations. The importance of these studies is highlighted by their possible impact on the development of novel therapeutic interventions. Potent small molecule activators of SIRT1 have recently become available with great promise in diseases associated with aging (Milne et al, Nature, 2007). We will use these activators to test the hypothesis that activating the suppressor function of SIRT1 will counterbalance the immune stimulatory function of Tat and may represent a novel strategy to treat chronic immune activation in HIV-infected patients. Our specific aims are: 1) we will study the role of SIRT1 in HIV-infected T cells. We will infect Jurkat T cell lines in which SIRT1 expression is down regulated via shRNAs with a GFP-tagged infectious clone of HIV-1 and study intracellular IL-2 production in infected (GFP+) and uninfected (GFP-) cells in response to activation with anti-CD3 and CD28 antibodies. We will also infect primary CD4+ T cell cultures with HIV-1 and test whether treatment with SIRT1 activators can reverse hyperstimulation of IL-2 production. 2) We will identify novel targets of the SIRT1 deacetylase activity in infected T cells. We have preliminary evidence that Tat by inhibiting SIRT1 induces hyperacetylation of several SIRT1 targets including proapoptotic factors p53 and Foxo3A. Since both factors are important regulators of T-cell death during HIV infection, we will study how Tat manipulates the transcriptional activities and proapoptotic functions of both factors. We will also perform genome-wide expression profiling and identify gene programs that are modulated by Tat expression or SIRT1 knockdown. 3) We will examine how the SIRT1/Tat interaction influences the development and function of Tregs. We have preliminary results showing that SIRT1 promotes TGF--induced Treg development and is involved in deacetylation of the transcription factor FoxP3. We propose to study how SIRT1 regulates expression and function of FoxP3 in Tregs and will characterize how Tat expression during HIV infection can interfere with this process. These studies will bring novel insight into the molecular biology of HIV-induced immune activation and identify yet undefined mechanisms of HDAC-mediated control of the immune response. PUBLIC HEALTH RELEVANCE: We seek to identify and characterize novel regulatory mechanisms controlling T cell hyperactivation during HIV-1 infection. T cell hyperactivation is a hallmark of pathogenic HIV-1 infection and the strongest predictor of the progression to AIDS in infected individuals. Our proposed studies characterize novel mechanisms by which the HIV-1 Tat protein hyperactivates T cells. These studies are directly relevant to HIV/AIDS and may contribute to the development of novel antiviral drugs that will address public need.